Practical Applications of DNA Testing

  • Italy is Keeping Cities Beautiful by DNA Testing Dog-Droppings
  • Fines Issued to Owners who Don’t Pick Up
  • Smart Idea or Story-plot for an Italian Comic Opera?

As you know, I like to explore all things related to nucleic acids. In this post, I take a little respite from scientific analysis and instead explore an interesting real-life application for DNA testing. As summer comes to an end and we’ve all returned from our recent holidays, this post provides a tie-in to Naples, which is both a great vacation destination and a place where dog-droppings are a controversial topic. Sound intriguing? Read on.

Naples’ historic city center, which is listed by UNESCO as a World Heritage Site, is the largest in Europe, covering 4,200 acres and enclosing 27 centuries (!) of history. This historic city is leading the way in employing modern technology to keep its streets clean. Naples is the first metropolitan city to use “CSI-style” DNA forensics to rid its sidewalks of dog poop and impose stiff fines to those who let Fido leave his DNA behind. It’s a story that strangely juxtaposes quaint European old-city charm with an interesting mix of science and societal topics to evoke a wide spectrum of opinions.

By the way, in researching Naples, I learned that it has long been a major cultural center with a global sphere of influence, particularly during the Renaissance and Enlightenment eras. In the immediate vicinity of Naples are numerous culturally and historically significant sites, including the Palace of Caserta and the Roman ruins of Pompeii and Herculaneum. Naples is synonymous with pizza, which originated in the city as Neapolitan flatbread and migrated to America with the Italians. Neapolitan music has furthermore been highly influential, credited with the invention of the romantic guitar and the mandolin, as well as notable contributions to opera.

Good Idea or Waste of a City’s Resources?

Despite its glorious past, the city is not without it’s slew of current problems. Tommaso Sodano, the vice mayor of Naples, recently acknowledged in an interview that the city is facing many challenges related to huge debt, unfunded service agencies, wide-spread organized crime and general filth related to illegal dumps and dog droppings…yes, poop.

The city administration is positioning Naples ‘at the cutting edge of dog-waste eradication’. The initiative takes DNA samples of dogs to create a database used to identify and fine owners who leave their dog’s poop on city streets.

“I know some people find it funny,” Mr. Sodano is quoted as saying, smiling, “that with all the problems the city has, we would focus on dog poop. I know that.”

Via Toledo is Naples’s principal shopping street and a “must do” for tourists (taken from Wikipedia)

Via Toledo is Naples’s principal shopping street and a “must do” for tourists (taken from Wikipedia)

While it may sound a bit humerous at first, it’s an issue that is wide-spread and difficult to control as there are some 80,000 dogs in Naples. While other cities have tried everything from mailing dog poop back to its owner to publicly shaming offenders, Naples is pursuing a more modern approach. Blood samples will be taken from every dog in the city and used to create a database matched with contact information for the dog’s owner. ‘When an offending pile is discovered, it will be scraped up and subjected to DNA testing. If a match is made in the database, the owner will face a fine of up to 500 euros, or about $685.’ The program is in its infancy, but officials say it’s already making an impact on Via Toledo and surrounding areas where it’s being piloted.

Veterinary workers in Naples drew blood from Fiona, a pit bull, for the DNA database. Credit Gianni Cipriano for the NY Times.

Veterinary workers in Naples drew blood from Fiona, a pit bull, for the DNA database. Credit Gianni Cipriano for the NY Times.

Many residents and officials are skeptical that the program will work given the city’s existing challenges with basic services like garbage collection and sewage. Others disagree with the program’s expenditures in light of the city’s mounting debt. Sodano remains committed to the program saying “The main goal is respect for the rules.” He’s quick to add that other mounting issues shouldn’t prevent city administration from keeping Naples beautiful. “Governing Naples,” he said, “certainly requires a sparkle of madness.”

What’s the DNA-Based ID Used?

I tried, unsuccessfully, to find out what DNA-based identity testing is being used in Naples. In researching this, however, I did find the abstract of a relatively recent publication that suggests to me that Naples may be analyzing mitochondrial DNA (mtDNA), analogous to TriLink’s forensic products (mitoPrimers™) for human identity testing using PCR-sequencing.

According to this abstract, the researchers sequenced the entire ∼16 kb canine mtDNA genome of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. They then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region sequence—identical in all dogs—for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. They identified a total of 71 informative SNPs that they concluded are “useful forensic tools to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).”

Even though we don’t know the exact details of the testing being used in Naples, I wrote this post because I find the practical applications of DNA-based testing to be fascinating. If you know of other real-life applications, please share them in the comments section below.

mtDNA Replacement: Eliminating Disease or Creating Designer Babies?

  • Mitochondrial DNA Replacement to Preclude Mitochondrial Disease Shown Feasible in Monkeys
  • US FDA Advisors Ponder Pros and Cons for Allowing Human Clinical Trials Amidst “Designer Baby” Concerns
  • UK Government Decision “Up in the Air”

Prologue

Before jumping into the headline and bylines of this post, I thought it would be worth sharing TriLink’s connection with mitochondrial DNA (mtDNA), as well as provide a bit of background about mtDNA and diseases related to it.

TriLink recently introduced its mitochondrial DNA (mtDNA) PCR-sequencing primers (mitoPrimers™) and mtDNA master mix for forensic science and casework. These products have been used by several well-known experts in mtDNA forensics, including Prof. Rhonda Roby, a well-known expert in mtDNA forensics. Prof. Roby’s expertise in mtDNA sequencing extends beyond forensic applications to academic interests in disease-related mtDNA dysfunction, as described in her recent publication in Nature Scientific Reports.

To better understand those interests, I researched mtDNA diseases at an NIH.gov website and found that, while mtDNA is only a “tiny” (~16,569 base-pairs) genetic component compared to genomic DNA (~2 billion base-pair), and encodes only 37 genes compared to ~20,000 genes for genomic DNA, the list of mtDNA related diseases is lengthy.

The vast majority (90%) of the energy needs of the human body are provided by mitochondrial oxidative phosphorylation that takes place entirely in mitochondria, and is a highly efficient system for producing the energy required to maintain the structure and function of the body. Consequently, according to the NIH.gov website, mutated mtDNA disrupts the mitochondria’s ability to efficiently generate energy for the cell, leading to organ-related health conditions. These conditions can be serious and are most pronounced in organs and tissues with high energy requirements, such as the heart, brain, and muscles. Frequently observed symptoms include muscle weakness and wasting, problems with movement, diabetes, kidney failure, heart disease, loss of intellectual functions (dementia), hearing loss, and abnormalities involving the eyes and vision.

mito

Taken from “Powerhouse Rules: The Role of Mitochondria in Human Diseases,” online coursework at MIT (ocw.mit.edu) via Bing Images.

The image (right) depicts multiple copies of circular mtDNA that reside in mitochondrial organelles external to a cell’s nucleus (in blue), which contains genomic DNA. Whether or not these organelles evolved by free-living bacteria that were taken inside a human cell-to-be (aka endosymbiotic theory) is an evolving—pun intended—debate worth reading about if you’re intrigued—as I am—by pondering how our cells came to be what they are.

Interestingly, women’s egg cells have many copies of mtDNA while men’s sperm cells have just enough to enable it to swim to the egg and fertilize it. The mtDNA of the sperm usually disappears once fertilization occurs. On the other hand, the mtDNA of the egg pass on to all of a woman’s children (male and female), but only women pass on their mtDNA from generation to generation. This maternal aspect of genetics is depicted in the cartoon below, posted by genetic-ancestry company 23andMe to celebrate Mother’s Day.

Taken from blog.23andme.com via Bing Images.

Taken from blog.23andme.com via Bing Images.

Unfortunately, we might not always be thanking Grandma for her mtDNA. In her daily blog, Mighty Mito Mom, Amy Boyd chronicles the challenges and experiences as a Mom whose daughter—affectionately called Little Miss Mollypop—has a mitrochondrial disease. I also found numerous YouTube videos that give a real sense of how families struggle with mitochondrial disease, such as this poignant example involving three generations affected by mitochondrial-related conditions. These examples raise the question, can something be done to preclude mtDNA disease by substituting “good mtDNA” for “bad mtDNA” during in vitro fertilization (IVF)?

The answer is yes, but the process involves what some view as three parents—two females and one male. As you may imagine, the procedure prompts considerable concern and controversy.

Mitalipov’s Mitochrondrial Manipulations

I’ve “borrowed” this catchy alliterative heading from the title of a March 2014 NY Times story. The article is by Sabrina Tavernise about Oregon Health and Science University Dr. Shoukhrat Mitalipov, who has developed a procedure to help women conceive children without passing on their mtDNA genetic defects. Salient snippets of this article are as follows, including this self-charactrization by Mitalipov: “my colleagues, they say I’m a ‘mitochondriac,’ that I only see this one thing. Maybe they are right.”

Dr. Mitalipov, 52, has shaken the field of genetics by perfecting a version of the world’s tiniest surgery: removing the nucleus from a human egg and placing it into another. In doing so, this Soviet-born scientist has drawn the ire of bioethicists and the scrutiny of federal regulators. Credit Leah Nash, NY Times.

Dr. Mitalipov, 52, has shaken the field of genetics by perfecting a version of the world’s tiniest surgery: removing the nucleus from a human egg and placing it into another. In doing so, this Soviet-born scientist has drawn the ire of bioethicists and the scrutiny of federal regulators. Credit Leah Nash, NY Times.

About one in 4,000 babies in the US is born with an inherited mitochondrial disease. These diseases are terminal, there is no known treatment, and few of these afflicted children live into adulthood. Women who carry mtDNA mutations are understandably eager not to pass them to their children. Remember, although sperm carry mitochondria, they are usually degraded shortly after fertilization, so mitochondrial diseases are passed down through the mother. Dr. Mitalipov’s procedure (shown below) would allow these women to bear children by placing the nucleus from the mother’s egg into a donor egg whose nucleus has been removed. The defective mitochondria, which float outside the nucleus in the egg’s cytoplasm, are left behind, thus eliminating the possibility of passing along defective mitochondria.

Those of you interested in Mitalipov’s published feasibility results with human oocytes can read a 2013 report in Nature, and a 2014 review of promises and challenges in Fertility and Sterility entitled “Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases.”

Mitochondrial DNA replacement could help carriers of severe disease have healthy children. Credit K. Sutliff, Science.

Mitochondrial DNA replacement could help carriers of severe disease have healthy children. Credit K. Sutliff, Science.

Are “Designer Babies” on the Horizon?

While some advocates of this procedure say it’s a “major breakthrough,”others aren’t as enthusiastic since the resulting baby would carry genetic material from three parents—the mother, the host egg’s donor and the father—an outcome that ethicists have deplored.

That specter drew critics from all over the country to a hotel in suburban Maryland in February 2014. It was here that Dr. Mitalipov tried to persuade a panel of experts convened by the FDA that the procedure, which he has pioneered in monkeys, was ready to test in people.

Some experts voiced concerns about unintended consequences, such as introducing new genetic mutations into the human gene pool. Others warned that it could be used later for something ethically murkier—perhaps, said Marcy Darnovsky, executive director of the Center for Genetics and Society, “to engineer children with specific character traits.”

Darnovsky’s assertion of a slippery slope situation possibly leading to “designer babies” is not new, and can be read about in a lengthy Science perspective entitled Stirring the Simmering “Designer Baby” Pot written by Thomas H. Murray. The metaphorical “pot” that he refers to ties Mitalipov’s proposed procedure to a 2013 patent granted to 23andMe—a controversial want-to-be genetic testing company currently foiled by the FDA. This “hot button” patent covers genetic calculations that enable would-be parents “to select a donor and view other possible phenotype of the hypothetical child resulting from the recipient’s and the donor’s gametes.”

Dr. Mitalipov waved off those warnings, according to Tavernise, by noting that mtDNA comprises just 37 genes, which direct the production of enzymes and molecules that the cell needs for energy. Those genes, he says, have nothing to do with traits like eye and hair color, which are encoded in the nucleus. She quotes Mitalipov as saying that “there are always people trying to stir things up. Many of them made their careers by criticizing me.”

“Suspended Animation”

In his Science perspective, Murray observes that “it is unrealistic to expect the US policy lacuna [gap or missing piece] over assisted reproductive technologies to be filled any time soon. Regulation of preimplantation genetic diagnosis remains in a ‘state of suspended animation’—according to a cited publication.

He adds that “professional self-regulation likely works well when the interested professions reflect a well-established public consensus. But when it comes to empowering parents to decide what sort of children they have beyond questions of serious childhood diseases, professional organizations cannot even agree on the appropriate ethical framework.”

Murray concludes by stating that “discussion of the ethics of mitochondrial manipulation cannot be postponed indefinitely. With little prospect of sensible legislation in the near term, and conflicting guidance from professional organizations, a national conversation about current and emerging technologies shaping the choices that parents will have is urgent. The UK conducted a similar exercise a decade ago that combined polling, focus groups, and the Internet.”

Status of mtDNA Replacement in the UK

In Britain, the government has issued draft regulations that would govern clinical trials of mtDNA replacement in people. If accepted into law by Parliament, such trials (which are now banned), would be allowed to go forward, although regulators would have to license any clinical application.

Polly Toynbee of The Guardian reported in February 2014 that, while a year has passed since the public was consulted at extraordinary length on the ethics of mtDNA replacement to prevent the birth of children with incurable genetic diseases—with most of the public saying yes, go ahead—the government has “dragged its feet.”

Inquiring about its progress with the Department of Health a couple of weeks before drafting her report, the answer was, “it’s up in the air.” Pressed on the question the day before publishing, she was told that draft regulations may appear next month. But the public will have to be consulted again, for a further three-month period. Polly opined that, since this procedure “arouses deep passions, plentiful responses are expected” and it will take yet more months to consider them. Doubts remain about the government’s eagerness to push this through parliament, rousing a controversy close to the general election, she added.

My comment is that difficult policy decisions and political party concerns for reelection seem to be similar on “both sides of the pond,” as the UK and the US are referred to.

Newcastle University neurologist Prof. Doug Turnbull, who is co-leading investigations with embryologist Dr. Mary Herbert, has been putting pressure on the government to prepare legislation that will allow experts in Newcastle to use human embryos containing DNA from three people to be used in clinical treatment, according to an earlier report.

Ms. Toynbee points out that the Human Fertilization and Embryology Authority (HFEA) conducted its scientific review of the procedure’s safety and efficacy and concluded back in 2011 that it would be unethical for the government not to press ahead, to prevent any more needless suffering. Toynbee was a member of the HFEA’s oversight group that supervised a massive public consultation, ensuring these complex issues were fairly aired and comprehensible to all.

Overall, the public was in favor, she says, and adds that when randomly selected people looked at the evidence they didn’t think this was a slippery slope that would lead to “designer babies,” or that it amounted to “three-parent IVF,” as there is no genetic effect on identity. The HFEA recommends that mitochondria are treated like tissue, a kidney for example—so donors would not be considered ‘parents.’

She adds “parliament is often less rational than the public. Stuffed with the religious and rabble-rousers who stir up fears of Frankenstein babies, many in both houses will make noisy speeches, ignoring the science.” That despite an estimated 73 people dying each year from mitochondrial disease, many of them children. The impact is even greater when you consider that some mitochondrial diseases go undiagnosed and that over 2,500 women of child-bearing age carry faulty genes – putting their children at risk.

Ms. Toynbee concluded her story by saying that “just as I reach the end of writing this, a call comes from the Health Department: it wants me to know that it is still absolutely committed, and regulations will be out soon. Why the year’s delay? Ah, um. When will the regulations go to parliament? ‘By the end of the year’ was the reply. Left so close to the election, let’s hope No. 10 strategists don’t veto it.”

As always, your comments are welcomed.

Postscript

After writing this blog, The New York Times Magazine featured a lengthy cover story by Kim Tingley entitled One Child, Three Parents. Also, a lengthy and freely available article by Ewen Callaway was published in venerable Nature magazine. Overall, the same pro and con issues are presented by Callaway, along with simplified diagrams showing methodological differences between pronuclear transfer and maternal spindle transfer techniques for genome transfer to prevent children from inheriting their mother’s mutant mitochondria. I found this article’s lead-in graphic (credited to Vasava) show below to be symbolically riveting and worth sharing here.

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